Method of increasing growth and yield in plants

ABSTRACT

Provided is a method of producing a genetically modified plant characterized as having increased growth and yield as compared to a corresponding wild-type plant comprising increasing the level of cyclin expression in the plant. The methods include providing a modified nucleic acid regulatory sequence from cycB1a;At resulting in increased gene transcription and expression. Also provided are modified nucleic acid regulatory sequences. Genetically modified plants characterized as having increased growth and yield are also provided.

[0001] This application is a continuation-in-part to U.S. applicationSer. No. 08/683,242, filed Jul. 18, 1996, the disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to plant geneticengineering, and specifically to a method for producing geneticallyengineered plants characterized as having increased growth and yield.

BACKGROUND OF THE INVENTION

[0003] For each plant species, there exists a wide discrepancy in plantgrowth due to environmental conditions. Under most conditions, themaximum growth potential of a plant is not realized. Plant breeding hasdemonstrated that a plant's resources can be redirected to individualorgans to enhance growth.

[0004] Genetic engineering of plants, which entails the isolation andmanipulation of genetic material, e.g., DNA or RNA, and the subsequentintroduction of that material into a plant or plant cells, has changedplant breeding and agriculture considerably over recent years. Increasedcrop food values, higher yields, feed value, reduced production costs,pest resistance, stress tolerance, drought resistance, the production ofpharmaceuticals, chemicals and biological molecules as well as otherbeneficial traits are all potentially achievable through geneticengineering techniques.

[0005] Plant growth responds to the increased availability of mineralnutrients in the soil, but shoot and root growth respond differently.Moreover, a direct relationship between mineral nutrient availabilityand change of growth rate is rarely observed over a larger concentrationrange. This suggest that plant growth is limited materially by nutrientsrequired for cell growth as well as by signaling pathways that controlthe rate of organ growth for the overall benefit of the plant. Althoughthe components of these regulatory pathways have not been identified,they define two distinct avenues to potentially improve plant growth. Ithas been shown that enhanced accumulation of cyclin protein undercontrol of the cdc2 promoter suffices to enhance root and overall plantgrowth under non-limiting conditions on growth media.

[0006] Plants rarely grow under optimal conditions. Plant growth can belimited by water availability, mineral nutrients and a short growingseason. Drought tolerance in genetic variants of a given species is wellcorrelated with the penetration depth of its root system into the soil.Fertilizers are often not optimally utilized because of insufficientlypenetrating root systems. Although the induction of flowering can now becontrolled, thereby extending the potential growth range of someimportant crop species, this does not in itself lead to increasedbiomass.

[0007] The ability to manipulate gene expression provides a means ofproducing new characteristics in transformed plants. For example, theability to increase the size of a plant's root system would permitincreased nutrient assimilation from the soil. Moreover, the ability toincrease leaf growth would increase the capacity of a plant toassimilate solar energy. Obviously, the ability to control the growth ofan entire plant, or specific target organs thereof would be verydesirable.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the discovery that increasedgrowth and yield in plants can be achieved by elevating the level ofcyclin expression.

[0009] In a first embodiment, the invention provides a method ofproducing a genetically modified plant characterized as having increasedgrowth and yield as compared to the corresponding wild-type plant. Themethod includes contacting a plant cell with a nucleic acid sequencecomprising a regulatory sequence, wherein said regulatory sequence isoperably associated with a nucleic acid encoding a cyclin protein, toobtain a transformed plant cell; producing plants from said transformedplant cell; and selecting a plant exhibiting said increased yield. Inparticular, the nucleic acid can be a cyclin gene and the regulatorysequence can be a cyclin gene promoter, such as cycB1a;At.

[0010] In another embodiment, the invention provides a transformed plantcell or a transformed plant having a nucleic acid sequence having aregulatory sequence of cycB1a;At operably linked to a heterologousnucleic acid sequence.

[0011] In yet another embodiment, the invention provides an isolatednucleic acid sequence having a functional cycB1a;At regulatory sequence.In a particular aspect, the regulatory sequence is the sequence setforth in SEQ ID NO:1.

[0012] The invention also provides plants, plant tissue and seedsproduced by the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0013]FIG. 1 shows steady state levels of cdc2aAt mRNA and p34 protein,panel a; cyc1aAt mRNA during IAA induction of lateral root meristems,panel b; cyc1aAt mRNA in selected non-induced transgenic lines, panel c;normalized transcript levels relative to wild-type are indicated. Col-0,wild-type; 1A2, 2A5, 4A3, 11A1: T2 homozygous; 6A, 7A, 8A: T1heterozygous transgenic lines.

[0014]FIG. 2 shows an in situ hybridization analysis of cdc2aAt andcyc1aAt transcripts in root apices and developing lateral roots. Panelsa-d show cross sections of quiescent roots (panels a,b) or proliferatingcells in primordia (panels c,d) that were hybridized to cdc2aAt (a) orcyc1aAt (b-d) anti-sense probes. Panels e, f show cyc1aAt mRNA abundancein contiguous meristematic cell files in root apices. Transcriptaccumulation is indicated by silver grain deposition and visualized byindirect red illumination. Scale bar is 10 μm in a-d, 5 μm in e. fc,founder cell accumulating cyc1aAt transcripts; p, pericycle cell layer;r, towards the root apex; s, towards the shoot.

[0015]FIG. 3 shows increased root growth rate in Arabidopsis thaliana(A. thaliana) ectopically expressing cyc1aAt cyclin. Panel a, Wild-type(left) or transgenic line 6A (T1 generation) containing thecdc2aAt::cyc1aAt gene fusion (right). Arabidopsis seed were plated on MS(3% sucrose) agar and grown in a vertical orientation for 7 d. Plantstransformed with the vector alone or with unrelated promoter::uidAconstructs or with a cdc2aAt::cyc1aAt fusion in which the cdc2aAt 5′untranslated leader was interrupted by a DS transposon insertion did notshow this phenotype. Panel b, wild-type (left) or transgenic line 6A (T1generation) (right) 6 d after IAA induction of lateral roots. Oneweek-old seedlings grown hydroponically were treated with 10 μMIAA_(eff) to stimulate lateral root development.

[0016]FIG. 4 shows aphidicolin (APC) release of the −1148 transformantand the −205 transformant.

[0017]FIG. 5 shows a series of constructs operably linked to cyc1a andGUS wherein a series of nested deletions were generated at the 5′ end ofthe cycB1a;At regulatory sequence.

[0018]FIG. 6 shows a Northern blot analysis that shows elevation ofendogenous levels of cyclins following treatment of roots with thegrowth-stimulatory hormone auxin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention provides methods for increasing the yieldof a plant, such as a agricultural crop, by elevating the cyclinexpression level in the plant. Increased cyclin expression in plantcells competent to divide results in increased plant growth.

[0020] In a preferred embodiment, the invention provides a method forproducing a genetically modified plant characterized as having increasedyield as compared to a plant which has not been genetically modified(e.g., a wild-type plant). The method comprises contacting plant cellswith nucleic acid encoding a cyclin protein, wherein the nucleic acid isoperably associated with a regulatory sequence to obtain transformedplant cells; producing plants from the transformed plant cells; andthereafter selecting a plant exhibiting increased growth and yield.

[0021] In a further embodiment, the regulatory sequence of the inventionis derived from a cycB1a;At gene. The regulatory sequence of thecycB1a;At gene is approximately 1.2kb in length. However, functionalfragments of this regulatory sequence are provided which confer amodified transcriptional activity upon nucleic acid sequence which areoperably linked to the regulatory sequence. By “modified transcriptionalactivity” is meant transcription of linked sequences above or belowwild-type expression of the linked sequence.

[0022] As used herein, the term “yield” or “plant yield” refers toincreased crop growth, and/or increased biomass. In one embodiment,increased yield results from increased growth rate and increased rootsize. In another embodiment, increased yield is derived from shootgrowth. In still another embodiment, increased yield is derived fromfruit growth.

[0023] As used herein, the term “agronomic” includes, but is not limitedto, changes in plant yield, growth or root size. Other agronomicproperties include insect resistance, protein production, droughttolerance, and other factors desirable to agricultural production andbusiness.

[0024] The term “genetic modification” as used herein refers to theintroduction of one or more exogenous nucleic acid sequences, e.g.,cyclin encoding sequences, as well as regulatory sequences, into one ormore plant cells, which can generate whole, sexually competent, viableplants. The term “genetically modified” as used herein refers to a plantwhich has been generated through the aforementioned process. Geneticallymodified plants of the invention are capable of self-pollinating orcross-pollinating with other plants of the same species so that theforeign gene, carried in the germ line, can be inserted into or bredinto agriculturally useful plant varieties. The term “plant cell” asused herein refers to protoplasts, gamete producing cells, and cellswhich regenerate into whole plants.

[0025] As used herein, the term “plant” refers to either a whole plant,a plant part, a plant cell, or a group of plant cells, such as planttissue or plant seed. Plantlets are also included within the meaning of“plant”. Plants included in the invention are any plants amenable totransformation techniques, including gymnosperms and angiosperms, bothmonocotyledons and dicotyledons.

[0026] Examples of monocotyledonous angiosperms include, but are notlimited to, asparagus, field and sweet corn, barley, wheat, rice,sorghum, onion, pearl millet, rye and oats and other cereal grains.Examples of dicotyledonous angiosperms include, but are not limited totomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers,lettuce, peas, alfalfa, clover, cole crops or Brassica oleracea (e.g.,cabbage, broccoli, cauliflower, brussel sprouts), radish, carrot, beets,eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers andvarious ornamentals. Examples of woody species include poplar, pine,sequoia, cedar, oak, etc.

[0027] The term “exogenous nucleic acid sequence” as used herein refersto a nucleic acid foreign to the recipient plant host or, native to thehost if the native nucleic acid is substantially modified from itsoriginal form. For example, the term includes a nucleic acid originatingin the host species, where such sequence is operably linked to apromoter that differs from the natural or wild-type promoter. In thebroad method of the invention, at least one nucleic acid sequenceencoding cyclin is operably linked with a promoter. It may be desirableto introduce more than one copy of cyclin polynucleotide into a plantfor enhanced cyclin expression. For example, multiple copies of a cyclinpolynucleotide would have the effect of increasing production of cyclineven further in the plant.

[0028] The term “regulatory sequence” as used herein refers to a nucleicacid sequence capable of controlling the transcription of an operablyassociated gene. Therefore, placing a gene under the regulatory controlof a promoter or a regulatory element means positioning the gene suchthat the expression of the gene is controlled by the regulatorysequence(s). In general, promoters are found positioned 5′ (upstream) ofthe genes that they control. Thus, in the construction of promoter genecombinations, the promoter is preferably positioned upstream of the geneand at a distance from the transcription start site that approximatesthe distance between the promoter and the gene it controls in thenatural setting. As is known in the art, some variation in this distancecan be tolerated without loss of promoter function. Similarly, thepreferred positioning of a regulatory element, such as an enhancer, withrespect to a heterologous gene placed under its control reflects itsnatural position relative to the structural gene it naturally regulates.

[0029] Cyclin-encoding nucleic acids utilized in the present inventioninclude nucleic acids encoding mitotic cyclins such as, for example,cyclin B; nucleic acids encoding S-phase cyclins such as, for examplecyclin A, and nucleic acids encoding G1 phase cyclins. Specific cyclinswhich can be utilized herein include cyc1aAt, cyc3aAt, cyc3bAt, cycB1a;At, cycd1, cycd2 and the like. Preferably, the nucleic acid used in themethod of the invention encodes the cyc1aAt protein (Genebank AccessionNo. X62279).

[0030] Genetically modified plants of the present invention are producedby contacting a plant cell with a nucleic acid sequence encoding thedesired cyclin. To be effective once introduced into plant cells, thecyclin-encoding nucleic acid must be operably associated with a promoterwhich is effective in plant cells to cause transcription of the cyclintransgene. Additionally, a polyadenylation sequence or transcriptioncontrol sequence, also recognized in plant cells, may also be employed.It is preferred that the nucleic acid be introduced via a vector andthat the vector harboring the nucleic acid sequence also contain one ormore selectable marker genes so that the transformed cells can beselected from non-transformed cells in culture, as described herein.

[0031] The term “operably associated” or “operably linked” refers tofunctional linkage between a regulatory sequence, preferably a promotersequence, and the cyclin-encoding nucleic acid sequence regulated by thepromoter. The operably linked promoter controls the expression of thecyclin nucleic acid sequence.

[0032] The expression of cyclin genes employed in the present inventionmay be driven by a number of promoters. Although the endogenous, ornative promoter of a structural gene of interest may be utilized fortranscriptional regulation of the gene, preferably, the promoter is aforeign regulatory sequence.

[0033] Such regulatory sequences include the cycB1a;At regulatorysequence and fragments thereof. Such fragments include sequences about−1148 bases upstream of the transcriptional start site (i.e., −1), aswell as sequence from −1 to −60, −1 to −120, −1 to −205, −1 to −286, and−1 to −351. Modified regulatory sequences thus provides a method todeliver increased levels of a desired gene of interest by modifying, forexample, repressor functions and enhancer functions orthe regulatorysequence. For example, deletions of the cycB1a;At regulatory sequencesuch that repressor functions are removed can provide a regulatorysequences which results in increased gene expression. Such deletions ofthe cycB1a;At sequence include, but are not limited to, cyclin promotersdeleted to −286 or −205.

[0034] When it is desired to increase growth and yield in the wholeplant, cyclin expression should be directed to all cells in the plantwhich are capable of dividing. This can be accomplished by using apromoter active in all meristems. Such promoters include, for example,the cdc2a promoter and the cyc07 promoter. (See for example, Ito et al.,Plant Mol. Biol., 24:863, 1994; Martinez et al., Proc. Natl. Acad. Sci.USA, 89:7360, 1992; Medford et al., Plant Cell, 3:359, 1991; Terada etal., Plant Journal, 3:241, 1993; Wissenbach et al., Plant Journal,4:411, 1993).

[0035] When it is desired to increase growth and yield in a specificorgan, cyclin expression should be targeted to the appropriate meristem,e.g., the shoot meristem, the floral meristem, the root meristem etc.This can be accomplished by using a tissue specific promoter. Examplesof tissue specific promoters active in shoot meristems are described inAtanassova et al., Plant Journal, 2:291, 1992 and Medford et al., PlantCell, 3:359, 1991. Examples of tissue specific promoters active infloral meristems are the promoters of the agamous and apetala 1 genesare described in Bowman et al., Plant Cell, 3:749, 1991; and Mandel etal., Nature, 360:273, 1992.

[0036] The particular promoter selected should be capable of causingsufficient cyclin expression to cause increased yield and/or increasedbiomass. It should be understood that cyclin expression can be alteredin cells that are competent to divide. The promoters used in the vectorconstructs of the present invention may be modified, if desired, toaffect their control characteristics. For example, deletions from the 5′end of the cycB1a;At regulatory sequence increases transcriptionalactivity.

[0037] Optionally, a selectable marker may be associated with thecyclin-encoding nucleic acid. As used herein, the term “marker” refersto a gene encoding a trait or a phenotype which permits the selectionof, or the screening for, a plant or plant cell containing the marker.Preferably, the marker gene is an antibiotic resistance gene whereby theappropriate antibiotic can be used to select for transformed cells fromamong cells that are not transformed. Examples of suitable selectablemarkers include adenosine deaminase, dihydrofolate reductase,hygromycin-B-phosphotransferase, thymidine kinase, xanthine-guaninephospho-ribosyltransferase and amino-glycoside 3′-O-phosphotransferaseII. Other suitable markers will be known to those of skill in the art.

[0038] To commence a transformation process in accordance with thepresent invention, it is first necessary to construct a suitable vectorand properly introduce it into the plant cell. Vector(s) employed in thepresent invention for transformation of a plant cell include acyclin-encoding nucleic acid sequence operably associated with apromoter, such as cycB1a;At or fragments thereof. Details of theconstruction of vectors utilized herein are known to those skilled inthe art of plant genetic engineering.

[0039] For plant expression vectors, suitable viral promoters includethe 35S RNA and 19S RNA promoters of CaMV (Brisson, et al., Nature,310:511, 1984; Odell, et al., Nature, 313:810, 1985); the full-lengthtranscript promoter from Figwort Mosaic Virus (FMV) (Gowda, et al., J.Cell Biochem., 13D: 301, 1989) and the coat protein promoter to TMV(Takamatsu, et al., EMBO J. 6:307, 1987). Alternatively, plant promoterssuch as the light-inducible promoter from the small subunit of ribulosebis-phosphate carboxylase (ssRUBISCO) (Coruzzi, et al., EMBO J., 3:1671,1984; Broglie, et al., Science, 224:838, 1984); mannopine synthasepromoter (Velten, et al., EMBO J., 3:2723, 1984) nopaline synthase (NOS)and octopine synthase (OCS) promoters (carried on tumor-inducingplasmids of Agrobacterium tumefaciens) or heat shock promoters, e.g.,soybean hsp17.5-E or hsp17.3-B (Gurley, et al., Mol. Cell. Biol., 6:559,1986; Severin, et al., Plant Mol. Biol., 15:827, 1990) may be used.

[0040] Promoters useful in the invention include both naturalconstitutive and inducible promoters as well as engineered promoters.The CaMV promoters are examples of constitutive promoters. To be mostuseful, an inducible promoter should 1) provide low expression in theabsence of the inducer; 2) provide high expression in the presence ofthe inducer; 3) use an induction scheme that does not interfere with thenormal physiology of the plant; and 4) have no effect on the expressionof other genes. Examples of inducible promoters useful in plants includethose induced by chemical means, such as the yeast metallothioneinpromoter which is activated by copper ions (Mett, et al., Proc. Natl.Acad. Sci., U.S.A., 90:4567, 1993); In2-1 and In2-2 regulator sequenceswhich are activated by substituted benzenesulfonamides, e.g., herbicidesafeners (Hershey, et al., Plant Mol. Biol., 17:679, 1991); and the GREregulatory sequences which are induced by glucocorticoids (Schena, etal., Proc. Natl. Acad Sci., U.S.A., 88:10421, 1991). Other promoters,both constitutive and inducible will be known to those of skill in theart.

[0041] The particular promoter selected should be capable of causingsufficient expression to result in the production of an effective amountof structural gene product, e.g., CDR1 polypeptide to cause increasedresistance to plant pathogens. The promoters used in the vectorconstructs of the present invention may be modified, if desired, toaffect their control characteristics.

[0042] Tissue specific promoters may also be utilized in the presentinvention. An example of a tissue specific promoter is the promoteractive in shoot meristems (Atanassova, et al., Plant J., 2:291, 1992).Other tissue specific promoters useful in transgenic plants, includingthe cdc2a promoter and cyc07 promoter, will be known to those of skillin the art. (See for example, Ito, et al., Plant Mol. Biol, 24:863,1994; Martinez, et al., Proc. Natl. Acad Sci. USA, 89:7360, 1992;Medford, et al., Plant Cell, 3:359, 1991; Terada, et al., Plant Journal,3:241, 1993; Wissenbach, et al., Plant Journal, 4:411, 1993).

[0043] There are promoters known which limit expression to particularplant parts or in response to particular stimuli. For example, potatotuber specific promoters, such as the patatin promoters or the promotersfor the large or small subunits of ADPglucose pyrophosphorylase, couldbe operably associated with CDR1 to provide expression primarily in thetuber and thus, provide resistance to attacks on the tuber, such as byErwinia. A fruit specific promoter would be desirable to impartresistance to Botrytis in strawberries or grapes. A root specificpromoter would be desirable to obtain expression of CDR1 in wheat orbarley roots to provide resistance to Ggt. One skilled in the art willknow of many such plant part-specific promoters which would be useful inthe present invention.

[0044] Alternatively, the promoters utilized may be selected to conferspecific expression of CDR1 in response to fungal infection. Theinfection of plants by fungal pathogens activate defense-related orpathogenesis-related (PR) genes which encode (1) enzymes involved inphenylpropanoid metabolism such as phenylalanine ammonia lyase, chalconesynthase, 4-coumarate coA ligase and coumaric acid 4-hydroxylase, (2)proteins that modify plant cell walls such as hydroxyproline-richglycoproteins, glycine-rich proteins, and peroxidases, (3) enzymes, suchas chitinases and glucanases, that degrade the fungal cell wall, (4)thaumatin-like proteins, or (5) proteins of as yet unknown function. Thedefense-related or PR genes have been isolated and characterized from anumber of plant species. The promoters of these genes may be used toobtain expression of CDR1 in transgenic plants when such plants arechallenged with a pathogen, particularly a fungal pathogen such as Pi.The particular promoter selected should be capable of causing sufficientexpression of CDR1 to result in the production of an effective amount ofpolypeptide.

[0045] Promoters used in the nucleic acid constructs of the presentinvention may be modified, if desired, to affect their controlcharacteristics. For example, the CaMV 35S promoter may be ligated tothe portion of the ssRUBISCO gene that represses the expression ofssRUBISCO in the absence of light, to create a promoter which is activein leaves but not in roots. The resulting chimeric promoter may be usedas described herein. For purposes of this description, the phrase “CaMV35S” promoter thus includes variations of CaMV 35S promoter, e.g.,promoters derived by means of ligation with operator regions, random orcontrolled mutagenesis, etc. Furthermore, the promoters may be alteredto contain multiple “enhancer sequences” to assist in elevating geneexpression.

[0046] Cyclin-encoding nucleic acid sequences utilized in the presentinvention can be introduced into plant cells using Ti plasmids ofAgrobacterium tumefaciens (A. tumefaciens), root-inducing (Ri) plasmidsof Agrobacterium rhizogenes (A. rhizogenes), and plant virus vectors.(For reviews of such techniques see, for example, Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, NY, SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9, and Horsch et al., Science, 227:1229,1985, both incorporated herein by reference). In addition to planttransformation vectors derived from the Ti or Ri plasmids ofAgrobacterium, alternative methods may involve, for example, the use ofliposomes, electroporation, chemicals that increase free DNA uptake,transformation using viruses or pollen and the use of microprojection.

[0047] One of skill in the art will be able to select an appropriatevector for introducing the cyclin-encoding nucleic acid sequence in arelatively intact state. Thus, any vector which will produce a plantcarying the introduced cyclin-encoding nucleic acid should besufficient. Even use of a naked piece of DNA would be expected to conferthe properties of this invention, though at low efficiency. Theselection of the vector, or whether to use a vector, is typically guidedby the method of transformation selected.

[0048] The transformation of plants in accordance with the invention maybe carried out in essentially any of the various ways known to thoseskilled in the art of plant molecular biology. (See, for example,Methods of Enzymology, Vol. 153, 1987, Wu and Grossman, Eds., AcademicPress, incorporated herein by reference). As used herein, the term“transformation” means alteration of the genotype of a host plant by theintroduction of cyclin-nucleic acid sequence.

[0049] For example, a cyclin-encoding nucleic acid can be introducedinto a plant cell utilizing A. tumefaciens containing the Ti plasmid, asmentioned briefly above. In using an A. tumefaciens culture as atransformation vehicle, it is most advantageous to use a non-oncogenicstrain of Agrobacterium as the vector carrier so that normalnon-oncogenic differentiation of the transformed tissues is possible. Itis also preferred that the Agrobacterium harbor a binary Ti plasmidsystem. Such a binary system comprises 1) a first Ti plasmid having avirulence region essential for the introduction of transfer DNA (T-DNA)into plants, and 2) a chimeric plasmid. The chimeric plasmid contains atleast one border region of the T-DNA region of a wild-type Ti plasmidflanking the nucleic acid to be transferred. Binary Ti plasmid systemshave been shown effective in the transformation of plant cells (DeFramond, Biotechnology, 1:262, 1983; Hoekema et al., Nature, 303:179,1983). Such a binary system is preferred because it does not requireintegration into the Ti plasmid of A. tumefaciens, which is an oldermethodology.

[0050] Methods involving the use of Agrobacterium in transformationaccording to the present invention include, but are not limited to: 1)co-cultivation of Agrobacterium with cultured isolated protoplasts; 2)transformation of plant cells or tissues with Agrobacterium; or 3)transformation of seeds, apices or meristems with Agrobacterium.

[0051] In addition, gene transfer can be accomplished by in plantatransformation by Agrobacterium, as described by Bechtold et al., (C.R.Acad. Sci. Paris, 316:1194, 1993) and exemplified in the Examplesherein. This approach is based on the vacuum infiltration of asuspension of Agrobacterium cells.

[0052] The preferred method of introducing a cyclin-encoding nucleicacid into plant cells is to infect such plant cells, an explant, ameristem or a seed, with transformed A. tumefaciens as described above.Under appropriate conditions known in the art, the transformed plantcells are grown to form shoots, roots, and develop further into plants.

[0053] Alternatively, cyclin-encoding nucleic acid can be introducedinto a plant cell using mechanical or chemical means. For example, thenucleic acid can be mechanically transferred into the plant cell bymicroinjection using a micropipette. Alternatively, the nucleic acid maybe transferred into the plant cell by using polyethylene glycol whichforms a precipitation complex with genetic material that is taken up bythe cell.

[0054] Cyclin-encoding nucleic acid can also be introduced into plantcells by electroporation (Fromm et al., Proc. Natl. Acad. Sci., U.S.A.,82:5824, 1985, which is incorporated herein by reference). In thistechnique, plant protoplasts are electroporated in the presence ofvectors or nucleic acids containing the relevant nucleic acid sequences.Electrical impulses of high field strength reversibly permeabilizemembranes allowing the introduction of nucleic acids. Electroporatedplant protoplasts reform the cell wall, divide and form a plant callus.Selection of the transformed plant cells with the transformed gene canbe accomplished using phenotypic markers as described herein.

[0055] Another method for introducing a cyclin-encoding nucleic acidinto a plant cell is high velocity ballistic penetration by smallparticles with the nucleic acid to be introduced contained either withinthe matrix of such particles, or on the surface thereof (Klein et al.,Nature 327:70, 1987). Bombardment transformation methods are alsodescribed in Sanford et al. (Techniques 3:3-16, 1991) and Klein et al.(Bio/Techniques 10:286, 1992). Although, typically only a singleintroduction of a new nucleic acid sequence is required, this methodparticularly provides for multiple introductions.

[0056] Cauliflower mosaic virus (CaMV) may also be used as a vector forintroducing nucleic acid into plant cells (U.S. Pat. No. 4,407,956).CaMV viral DNA genome is inserted into a parent bacterial plasmidcreating a recombinant DNA molecule which can be propagated in bacteria.After cloning, the recombinant plasmid again may be cloned and furthermodified by introduction of the desired nucleic acid sequence. Themodified viral portion of the recombinant plasmid is then excised fromthe parent bacterial plasmid, and used to inoculate the plant cells orplants.

[0057] As used herein, the term “contacting” refers to any means ofintroducing a cyclin-encoding nucleic acid into a plant cell, includingchemical and physical means as described above. Preferably, contactingrefers to introducing the nucleic acid or vector containing the nucleicacid into plant cells (including an explant, a meristem or a seed), viaA. tumefaciens transformed with the cyclin-encoding nucleic acid asdescribed above.

[0058] Normally, a plant cell is regenerated to obtain a whole plantfrom the transformation process. The immediate product of thetransformation is referred to as a “transgenote”. The term “growing” or“regeneration” as used herein means growing a whole plant from a plantcell, a group of plant cells, a plant part (including seeds), or a plantpiece (e.g., from a protoplast, callus, or tissue part).

[0059] Regeneration from protoplasts varies from species to species ofplants, but generally a suspension of protoplasts is first made. Incertain species, embryo formation can then be induced from theprotoplast suspension. The culture media will generally contain variousamino acids and hormones, necessary for growth and regeneration.Examples of hormones utilized include auxins and cytokinins. Efficientregeneration will depend on the medium, on the genotype, and on thehistory of the culture. If these variables are controlled, regenerationis reproducible.

[0060] Regeneration also occurs from plant callus, explants, organs orparts. Transformation can be performed in the context of organ or plantpart regeneration. (see Methods in Enzymology, Vol. 118 and Klee et al.,Annual Review of Plant Physiology, 38:467, 1987). Utilizing the leafdisk-transformation-regeneration method of Horsch et al., Science,227:1229, 1985, disks are cultured on selective media, followed by shootformation in about 2-4 weeks. Shoots that develop are excised from calliand transplanted to appropriate root-inducing selective medium. Rootedplantlets are transplanted to soil as soon as possible after rootsappear. The plantlets can be repotted as required, until reachingmaturity.

[0061] In vegetatively propagated crops, the mature transgenic plantsare propagated by utilizing cuttings or tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenotesis made and new varieties are obtained and propagated vegetatively forcommercial use.

[0062] In seed propagated crops, mature transgenic plants can be selfcrossed to produce a homozygous inbred plant. The resulting inbred plantproduces seed containing the newly introduced foreign gene(s). Theseseeds can be grown to produce plants that would produce the selectedphenotype, e.g. increased yield.

[0063] Parts obtained from regenerated plant, such as flowers, seeds,leaves, branches, roots, fruit, and the like are included in theinvention, provided that these parts comprise plant cells that have beentransformed as described. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

[0064] Plants exhibiting increased growth and/or yield as compared withwild-type plants can be selected by visual observation. The inventionincludes plants produced by the method of the invention, as well asplant tissue and seeds.

[0065] In another embodiment, the invention provides a method ofproducing a plant characterized as having increased growth and yield bycontacting a plant capable of increased yield with a cyclinpromoter-inducing amount of an agent which induces cyclin geneexpression. Induction of cyclin gene expression results in production ofa plant having increased yield as compared to a plant not contacted withthe agent.

[0066] A “plant capable of increased yield” refers to a plant that canbe induced to express its endogenous cyclin gene to achieve increasedyield. The term “promoter inducing amount” refers to that amount of anagent necessary to elevate cyclin gene expression above cyclinexpression in a plant cell not contacted with the agent, by stimulatingthe endogenous cyclin promoter. For example, a transcription factor or achemical agent may be used to elevate gene expression from native cyclinpromoter, thus inducing the promoter and cyclin gene expression.

[0067] The invention also provides a method of providing increasedtranscription of a nucleic acid sequence in a selected tissue. Themethod comprises growing a plant having integrated in its genome anucleic acid construct comprising, an exogeneous gene encoding a cyclinprotein, said gene operably associated with a tissue specific wherebytranscription of said gene is increased in said selected tissue.

[0068] Plant development is plastic with post-embryonic organogenesismediated by meristems (Steeves and Sussex, Patterns in PlantDevelopment, 1-388 (Press Syndicate of the University of Cambridge,Cambridge, 1989)). Although cell division is intrinsic to meristeminitiation, maintenance and proliferative growth, the role of the cellcycle in regulating growth and development is unclear. To address thisquestion, the expression of cdc2 and cyclin genes, which encode thecatalytic and regulatory subunits, respectively, of cyclin-dependentprotein kinases controlling cell cycle progression (Murray and Hunt, TheCell Cycle (New York), 1993) were examined. Unlike cdc2, which isexpressed not only in apical meristems but also in quiescent meristems,(Martinez et al., Proc. Natl. Acad. Sci. USA, 89:7360, 1992),transcripts of cyc1aAt accumulated specifically in active meristems anddividing cells immediately before cytokinesis. Ectopic expression ofcyc1aAt under control of the cdc2aAt promoter in Arabidopsis plantsmarkedly accelerated growth without altering the pattern of developmentor inducing neoplasia. Thus, cyclin expression is a limiting factor forgrowth.

[0069] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLE 1

[0070] A full length cyc1aAt cyclin cDNA was placed under control of theArabidopsis cdc2aAt promoter (Hemerly et al, 1992, supra). The chimericgene was cloned into a T-DNA transformation vector carrying theselection marker hygromycin phospho-transferase (Hyg′) and transformedinto Arabidopsis using the vacuum-infiltration method (Bechtold andPelletier, Acad. Sci. Paris, Life Sci., 316:1194, 1993) to introduceAgrobacterium tumefaciens. Several independent transgenic lines havingelevated steady-state levels of cyc1aAt mRNA showed a dramatic increaseof both main and lateral root growth rate, correlated withproportionally increased fresh weight, dry mass and DNA content, but notcell size. Enhanced growth was orderly, with no observed differences inmorphology and clearly not neoplastic.

[0071] Arabidopsis seedlings (ecotype Columbia) were grown in 20 ml MSmedium (Murashige and Skoog, Physiol. Plant., 15:473, 1962). Eight- to10-day-old plants were transferred to MS medium buffered with 50 mMpotassium phosphate, pH 5.5, and initiation of lateral roots wasstimulated by addition of IAA to 10 μM_(eff) (non-dissociated IAA).Roots were collected at the time points indicated and total RNA andprotein isolated. 500 ng poly(A)+ RNA was separated on 1% formaldehydegels (Ausubel et al., Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley-Interscience, New York, 1987),transferred to Nytran membranes (Schleicher and Schüll) and hybridizedto ³²P-labeled probes corresponding to nucleotides (nt) 674-1004 ofcyc1aAt (Hemerly et al., Proc. Natl. Acad. Sci. USA, 89:3295, 1992), ornt 661-1386 of Arabidopsis cdc2aAt (Hirayama et al., Gene, 105:159,1991), followed by hybridization with nt 2576-2824 of Arabidopsis UBQ3(Norris et al., Plant Mol. Biol., 21:895, 1993) for normalization. Blotswere quantified with a Molecular Dynamics Phosphorimager. cyc1aAt is asingle copy gene in Arabidopsis. Total protein was separated on 12%SDS-PAGE and transferred to PVDF membranes. p34^(cdc2aAt) was detectedwith serum raised in rabbits against the peptide YFKDLGGMP (SEQ IDNO:1), corresponding to amino acids 286-294, and visualized by EnhancedChemiluminescence Assay (Amersham).

[0072]FIG. 1 shows steady state levels of cdc2aAt mRNA and p34 protein,panel a; cyc1aAt mRNA during IAA induction of lateral root meristems,panel b; cyc1aAt mRNA in selected non-induced transgenic lines, panel c;normalized transcript levels relative to wild-type are indicated. Col-0,wild-type; 1A2, 2A5, 4A3, 11A1: T2 homozygous; 6A, 7A, 8A: T1heterozygous transgenic lines. cyc1aAt mRNA levels in the lines 4A3, 6A,7A, 8A, and 3A exceeded those of IAA induced wild-type roots.

[0073] The levels of cdc2 mRNA and p34^(cdc2) protein per cell did notmarkedly change following stimulation of lateral root initiation by theauxin indoleacetic acid (IAA) (FIG. 1, panel a). Hence, while cdc2expression is correlated with the competence to divide, root growth andinitiation of lateral roots do not appear to be limited by the abundanceof the cyclin-dependent protein kinase p34 catalytic subunit and,moreover, ectopic expression of cdc2 in transgenic Arabidopsis failed toperturb growth or development (Hemerly et al., EMBO J., 14:3925, 1995).

[0074] In contrast, IAA treatment of Arabidopsis roots induced theexpression of several cyc genes from low basal levels and in particularcyc1aAt mRNA, which encodes a mitotic cyclin (Hemerly et al., supra)),exhibited a rapid 15 to 20-fold increase (FIG. 1, panel b).

[0075]FIG. 2 shows an in situ hybridization analysis of cdc2aAt andcyc1aAt transcripts in root apices and developing lateral roots. Panelsa-d show cross sections of quiescent roots (panels a,b) or proliferatingcells in primordia (panels c,d) that were hybridized to cdc2aAt (a) orcyc1aAt (b-d) anti-sense probes. Panels e,f show cyc1aAt mRNA abundancein contiguous meristematic cell files in root apices. Transcriptaccumulation is indicated by silver grain deposition and visualized byindirect red illumination. Scale bar is 10 μm in a-d, 5 μm in e. fc,founder cell accumulating cyc1aAt transcripts; p, pericycle cell layer;r, towards the root apex; s, towards the shoot.

[0076] Tissue samples were processed for in situ hybridization toexamine expression of cyclin transcripts. The samples were treated with10 μM IAA. After 8 or 24 h incubation, radish (Raphanus sativa varScarlet Globe) roots were processed as described (Drews et al., Cell,65:991, 1991). Sections (8 μm) were hybridized to a ³³P-labeled RNAprobe, corresponding to nt 674-1004 of cyc1aAt (Hemerly et al., supra)(FIG. 2, panels b-e) or to a ³⁵S-labeled probe used in a correspondingto nt 661-1386 of cdc2aAt (Hirayama et al., supra), for 14 h at 48° C.in 50% formamide. After hybridization, the final washes were for 1 h at58° C. in 0.015 m NaCl and slides were then exposed for 3 weeks(cyc1aAt) or 5d (cdc2aAt). After developing, silver grains wereilluminated laterally with red light, specimens were visualized by phasecontrast and double exposures were taken on FUJI Velvia film. Imageswere assembled in ADOBE Photoshop. For the analysis summarized in FIG.2, panel f silver grains were counted and cell size measured in the cellfile shown in FIG. 2, panel e.

[0077] In situ hybridization showed that, unlike cdc2, cyc1aAttranscripts were not detected in quiescent pericycle cells, butaccumulated in single, cytoplasmically dense cells of incipient lateralroot primordia, and in the emergent organ cyc1aAt was expressedexclusively in the meristem (FIG. 2, panels a-d). Moreover, cruciferroots consisted of long cell files that arise by transverse divisionsfollowed by longitudinal expansion (Dolan et al., Development, 119:71,1993), and within such a contiguous spatial display of sequential celldivision phases, cyc1aAt transcripts accumulated only in large cellsimmediately prior to cytokinesis, declining to background levels in theadjacent small daughter cells (FIG. 2, panels e,f). A similar, stringentspatio-temporal relationship of cyclin expression and mitosis wasobserved in Antirrhinum shoot apical meristems (Fobert et al., EMBO J.,13:616, 1994).

[0078] The close correlation between cyc1aAt expression and celldivision during growth of the root apical meristem and the initiation oflateral roots, together with the pattern of cyc1aAt promoter activitydeduced from the expression of cyc1aAt::uidA gene fusions in transgenicArabidopsis (Ferreira et al., Plant Cell, 6:1763, 1994), suggested thatcyclin abundance might be a key factor regulating root growth anddevelopment. To test this hypothesis transgenic Arabidopsis weregenerated (Bechtold and Pelletier, Acad. Sci. Paris, Life Sci.,316:1194, 1993) containing cyc1aAt under control of the cdc2aAtpromoter. Five transformants were obtained in which the level of cyc1aAtmRNA in untreated roots exceeded that observed in IAA-stimulated rootsof wild-type plants (FIG. 1, panel c), and these lines were chosen forfurther study.

[0079] An NheI site was introduced in the third codon of the cyc1aAtcDNA by in vitro mutagenesis and this open reading frame subsequentlyligated to the cdc2aAt promoter with an in vitro generated XbaI site atcodon 3. This fragment was ligated into pBiB-Hyg (Becker et al., Pl.Mol. Biol., 20:1195, 1992) and transfected into Agrobacteriumtumefaciens GV3101 (Koncz and Schell, Mol. Gen. Genet., 204:383, 1986).Arabidopsis thaliana (A. thaliana) (ecotype Columbia) was transformed byvacuum infiltration (Bechtold et al., supra), and transgenic seedlings(T0 generation) were selected on MS plates containing 30 μg/mlhygromycin. 52 independent transgenic lines were obtained and elevatedlevels of cyc1aAt mRNA were detected in 9 of the 11 lines analyzed indetail. Growth assays were performed on heterozygous T1 and homozygousT2 progeny as indicated.

[0080]FIG. 3 shows increased root growth rate in Arabidopsis ectopicallyexpressing cyc1aAt cyclin. Panel a, Wild-type (left) or transgenic line6A (T1 generation) containing the cdc2aAt::cyc1aAt gene fusion (right).Arabidopsis seed were plated on MS (3% sucrose) agar and grown in avertical orientation for 7 d. Plants transformed with the vector aloneor with unrelated promoter::uidA constructs or with a cdc2aAt::cyc1aAtfusion in which the cdc2aAt 5′ untranslated leader was interrupted by aDS transposon insertion did not show this phenotype. Panel b, wild-type(left) or transgenic line 6A (T1 generation) (right) 6 d after IAAinduction of lateral roots. One week-old seedlings grown hydroponicallywere treated with 10 μM IAA_(eff) to stimulate lateral root development.

[0081] Strong expression of the cdc2aAt::cyc1aAt transgene caused amarked increase in the rate of organized root growth (FIG. 3, panel a).Homozygous or heterozygous seed were plated on MS agar and plants grownin vertical orientation for 7 days with a 16 h day/8 h night schedule at22° C. Four images of each plate were acquired with a SpeedlightPlatinum frame grabber (Lighttools Research) at 24 h intervals and rootgrowth analyzed with NIH-Image by measuring the displacement of rootapices. Following growth analysis, roots from 10 plants of each classwere collected and RNA analyzed. To measure cell sizes, roots werecleared by overnight incubation in saturated chloral hydrate, visualizedwith Normarski optics, photographed and analyzed with NIH-Image.Statistical analysis (t-test with unpaired variances) was performed withMS Excel. Root growth in IAA-treated plants was assessed 3 and 6 d afterinduction by determination of fresh weight of roots excised fromliquid-grown plants and then dry weight following lyophilization for 24h. Total DNA was extracted from dried material (Ausubel et al., supra).

[0082] In heterozygous T2 progeny, increased growth rate, measured bydisplacement of the apex of the main root in time-lapse photography,strictly co-segregated with transgene expression and individuals lackingthe transgene grew at the same rate as wild-type plants (Table 1). Theaverage size of epidermal, cortical, endodermal and pericycle cells wasequivalent or slightly reduced in cdc2aAt::cyc1aAt transfonnantscompared to wild-type plants (Table 2), and hence increased growthreflects increased cell number rather than cell size. The pattern ofspontaneous lateral root initiation and overall root morphology wereindistinguishable in wild-type and transgenic plants (FIG. 3, panel a).When treated with 1 μM IAA, which induces well-separated lateral rootprimordia, the frequency of primordia initiated per unit length of themain roots was not altered (mean of 1.08 initials/mm with a standarddeviation of 0.09 in wild-type compared with 1.14±0.07 and 1.09±0.13 inthe two transgenic lines examined). However, growth and development oflateral roots following induction by 10 μM IAA, was markedly acceleratedin the cdc2aAt::cyc1aAt transformants, giving rise to a much enlargedroot system (FIG. 3, panel b). Enhanced root growth in cdc2aAt::cyc1aAtplants following IAA treatment superficially resembles the alf1phenotype (Celenza et al., Genes & Development, 9:2131, 1995) and theseplants have elevated levels of cyc1aAt transcripts but in contrast tocdc2aAt::cyc1aAt transformants, alf1 plants initiate supernumerarylateral roots. The several-fold greater gain of fresh weight inIAA-treated cdc2aAt::cyc1aAt plants compared to equivalent wild-typecontrols was accompanied by marked increased in DNA content and dryweight (Table 3). Confocal microscopy confirmed that the enhanced growthresponse to IAA, which was also observed in several lines showing weakercdc2aAt::cyc1aAt expression, did not reflect transgene stimulation ofcell vacuolation or elongation. Thus, ectopic cyclin expression enhancesroot growth by stimulation of cell division activity in meristems,thereby increasing the rate of cell production without altering meristemorganization.

[0083] The data above indicate that cdc2aAt::cyc1aAt expression issufficient to enhance growth from established apical meristems,suggesting that cell cycle activity regulates meristem activity.However, the failure to induce gratuitous organ primordia by ectopicexpression of cyc1aAt under control of the cdc2aAt promoter impliesadditional control points in the generation of a new apical meristem,either through post-translational regulation of cyclin-dependent proteinkinase activity or the operation of parallel regulatory pathways. Inmost animal cells, the commitment to cell division occurs late in G1(Pardee, A. B., Science, 246:603, 1989), and cyclin D1 and cyclin E arerate-limiting for G1 progression in cultured cells (Ohtsubo and Roberts,Science, 259:1908, 1993; Quelle et al., Genes Dev., 7:1559, 1993;Resnitzky and Reed, Mol. Cell Biol., 15:3463, 1995). Elevated levels ofcyclin D1 are observed in several tumors (Motokura et al., Nature,350:512, 1991; Rosenberg et al., Proc. Natl. Acad. Sci. USA, 88:9638,1991; Withers et al., Mol. Cell Biol., 11:4864, 1991) and ectopicexpression in transgenic mice promotes hyperplasia and adenocarcinomas(Wang et al., Nature, 369:669, 1994).

[0084] In contrast, ectopic expression of cyc1aAt did not result inneoplasia but stimulated organized growth, without altering meristemorganization or size as monitored by confocal microscopy. Moreover,morphology of the transgenic plants was not altered and increased growthwas accompanied by accelerated organ development. Thus, cyclinexpression is a crucial, limiting upstream factor in an intrinsicregulatory hierarchy governing meristem activity, organized growth andindeterminate plant development. This regulatory hierarchy, which isdistinctly different from that in animals, where determinate developmentlimits proliferative growth, exemplified by the strict morphogeneticcontrol of cell division during muscle differentiation (Halevy et al.,Science, 267:1018, 1995; Skapek et al., Science, 267:1022, 1995), mayunderlie the striking plasticity of plant growth and development (Drew,M. C., New Phytol., 75:479, 1975). Cyclin abundance may function as arheostat to allow flexible growth control in response to changes in theenvironment such as nutrient availability. TABLE 1 Root apical growthPlant line Rate [μm · h⁻¹] % of wild-type n Col-0 (−) 254.1 100 57 2A5(−) 253.1 99.6 56 3A (+) 341.4* 134.4 20 3A (−) 259.4 102.1 30 4A3 (+)291.6* 114.8 47 6A (+) 354.1* 139.5 20 6A (−) 252.4 99.3 16 7A (+)344.9* 135.7 24 7A (−) 249.8 98.3 19 8A (+) 335.4* 131.9 21 8A (−) 258.6101.8 31 11A1 (−) 258.8 101.8 45 2A5 (−) 253.1 99.6 56

[0085] TABLE 2 Plant line Col-0 7A 8A (wild type) (transgenic)(transgenic) Cell Type Size [μm] n Size [μm] n Size [μm] n Epidermis 13737 129  34 158 12 Cortex 159 31 135*  7 160  9 Endodermis 109 23  90* 22107 11 Pericycle  73 26 67 19  71 57

[0086] TABLE 3 Growth of seedling root system Fresh weight Dry weightDNA per root [mg] [mg] [μg] Plant line 3d 6d 3d 6d 3d 6d Col-0 11  251.7  2.4 5 14 4A3 31 136 4.5 15.4 8 35 6A 30 155 4.2 19.3 10  46 7A 24156 3.5 16.8 9 38 8A 18 134 2.5 12.8 8 33

[0087] One aspect of cyclin overexpression in the CDC2a::cyc1aAttransgenic plants is its role in stimulating cyclin-dependent kinase(CDK) complex activity. Cyclins function to activate the catalyticsubunit of such complexes. Several lines of genetic evidence indicatethat specific cyclin genes largely differ in their regulation during thecell cycle, but not in their biochemical ability to activate kinaseactivity. This evidence indicates that structurally divergent cyclinscan functionally substitute for each other. First, the budding yeastgenome encodes for only one catalytic subunit of the cyclin-dependentkinase gene family (CDC28) involved in governing cell cycle progress.However, it encodes for at least 9 cyclins which are only distantlyrelated by structure but which nonetheless all activate CDK activityafter associating with the p34cdc28 protein encoded by CDC28. Second,Drosophila or human cyclin genes have been isolated by way ofcomplementation of a triple yeast G1 cyclin (CLN) knockout strain. Notonly were animal G1 cyclins isolated but mitotic cyclins were isolatedas well. Recent research has also shown that yeast CLB or mitotic typecyclins can bypass the requirement for CLN cyclins.

[0088] Taken together, these observations demonstrate that distinctlyregulated, and structurally only very distantly related cyclins (some ofthese share only 19% homology) substitute for one another such that theyare able to activate cyclin-dependent kinase activity by associationwith the catalytic subunit, albeit with varying degrees of efficiency.Thus, plant cyclins in general have the inherent property of enhancingroot and plant growth by virtue of their capacity to associate withendogenous proteins encoded by CDK homologous genes, and therefore thepresent invention applies to other plant cyclins beyond cyc1aAt.

[0089]FIG. 6 shows a Northern blot analysis that shows elevation ofendogenous levels of cyclins following treatment of roots with thegrowth-stimulatory hormone auxin. The blot shows that 5 differentmitotic cyclin genes and one G1 cyclin gene were all expressed atelevated levels upon auxin (IAA) treatment which in due course leads tothe initiation of a new root. The induction of many endogenous cyclingenes following growth stimulation leads to the conclusion that theregulatory pathways controlling growth in plants enhance cyclinexpression levels in general. Thus, cell division enhancement in thecourse of growth stimulation is not likely a unique property ofexpressing a specific cyclin gene or enhancing the level of its proteinproduct.

[0090] The emerging picture that cyclins exist in even larger genefamilies in plants than in yeast or animals is not unexpected (Renaudinet al. Plant Mol. Biol. [1996] 32 1003-1018). Regulatory genes in plantstend to exist in large, distinctly regulated gene families. For example,the regulatory genes controlling pigment (anthocyanin) synthesisconstitute gene families. Most of the bright red and blue colors foundin higher plants are anthocyanins. Using corn as an example for the restof the plant world, crucial regulatory genes of the helix-loop-helixclass (R, B, Lc) and of the myb class (C1, P1) are required to stimulatetranscription of the biosynthetic genes of the anthocyanin pathway.These regulatory genes are functionally redundant, in other words theycan replace each other. However in maize, they are regulateddifferentially, such that P1 is required for anthocyanin pigmentation inthe vegetative tissue of the plant, and is sensitive to light intensity,whereas C1 operates in reproductive tissues and operates constitutively.Similar properties hold for the R, B, Lc family. Constitutive expressionof the maize R-gene suffices to induce ubiquitous anthocyaninbiosynthetic genes and pigment accumulation in the heterologous plantArabidopsis (Lloyd A M et al. Science, [1992] 258 1773-1775).

[0091] Another example for complex regulatory gene families in plantsare the members of the calmodulin-like domain protein kinase gene family(Hrabak et al. Plant Mol. Biol. [1996] 31 405-412). At least 12 suchgenes are now known in Arabidopsis. These enzymes are all activated bycalcium, but their expression patterns in the plant are quite distinct.

[0092] Taken together, plants have evolved complex multi-gene familiesof regulatory genes that are functionally, i.e. biochemically,redundant, but differentially regulated. Thus, structurally divergentmembers of gene families combine a common biochemical activity withdifferential regulation likely to enable the plant to appropriatelyrespond to environmental and developmental cues.

EXAMPLE 2

[0093] A 2.8 kb HindIII fragment including approximately 1.2 kb ofupstream sequence and genomic sequence corresponding to the first 306amino acids encoded by the cycB1a;At gene was isolated. 3′ deletions ofthis fragment were generated by exonuclease III treatment and a 1.8 kbfragment that included promoter sequence and coding sequence up to aminoacid 116 was selected to generate a translational fusion toβ-glucuronidase (GUS). Subsequently, a series of nested 5′ deletions wasgenerated. The plasmids described here terminated at 351, 286, 205, 120,60 and 9 bp, respectively, upstream of the transcription start site. Theresulting series of constructs (FIG. 5) were cloned into the KpnI-SacIsites of the pBIB transformation vector (Becker et al., (1990), Nucl.Acids Res. 18:203) and subsequently introduced into tobacco BY2 cellsand Arabidopsis plants by Agrobacterium-medicated transformation (An,G., (1985), Plant physiol., 568-570; Bechtold et al., (1993), C.R. Acad.Sci. Paris, Life Sci. 316:1194-1199).

[0094] To establish a synchronized cell population, N. Tabacum BY2(pCDG) cells were arrested in S phase with the DNA polymerase inhibitorAphidicolin (APC) (Kodama and Komamine, (1995), Meth. Cell Biol.49:315-329). Following release from cell cycle arrest, samples werewithdrawn every hour for RNA analysis. In cells transformed with the−1148 or −351 cyclin promoter-GUS construct, RNA abundance increasedapproximately 50-fold as cells entered M-phase. However, in cellstransformed with cyclin promoters deleted to −286 or −205, RNA abundanceincreased approximately 200-fold as cells entered M-phase (FIG. 4).Further removal of 5′ sequences substantially reduced the amplitude ofcyclin induction (−120, −60 deletions), until cell cycle-regulatedexpression was lost after deletion to −9. Therefore, the DNA sequencebetween −351 and −286 comprises a binding motif for a cognate proteinwith repressor function, the sequence between −205 and −60 comprises anactivator motif and sequences between −60 and −9 are responsible forcell cycle regulation. Utilization of a cyclin promoter deleted to −286or −205 therefore provides a method to deliver increased levels of adesired gene of interest specifically at G2/M.

[0095] The XhoI site present within the third cyclin exon was use toexchange the E. coli UidA coding sequence with the remainder of thecyclin cDNA. A series of constructs with 5′-deleted promoters was usedto transform Arabidopsis and evaluate the growth properties of theresultant plants. Nine out of 13 independent transgenic lines that haveelevated steady state levels of cycB1a;At mRNA show a 10-25% increase ofroot growth rate, as well as increased fresh weight and dry mass but nocell size. When grown on soil, overall plant growth, specifically therate of leaf growth is dramatically enhanced, although neither finalsize of the plant nor flowering time are strongly affected. Thus,increased growth early during leaf development results in a cumulativepositive effect on plant growth, presumably because photosynthate can besupplied to sink tissues earlier than in non-transformed controls.Enhanced growth is orderly, with no differences in organ morphology andwith no evidence for neo- or hyperplasia.

[0096] The foregoing description of the invention is exemplary forpurposes of illustration and explanation. It should be understood thatvarious modifications can be made without departing from the spirit andscope of the invention. Accordingly, the following claims are intendedto be interpreted to embrace all such modifications.

1. A method of producing a genetically modified plant characterized ashaving increased growth and yield as compared to the correspondingwild-type plant, said method comprising: contacting a plant cell with anucleic acid sequence comprising a regulatory sequence, wherein saidregulatory sequence is operably associated with a nucleic acid encodinga cyclin protein, to obtain a transformed plant cell; producing plantsfrom said transformed plant cell; and selecting a plant exhibiting saidincreased yield.
 2. The method of claim 1 , wherein the geneticallymodified plant exhibits increased root growth.
 3. The method of claim 1, wherein the genetically modified plant exhibits increased shootgrowth.
 4. The method of claim 1 , wherein the cyclin is cyc1aAt.
 5. Themethod of claim 1 , wherein the regulatory sequence comprises acycB1a;At promoter.
 6. The method of claim 5 , wherein the regulatorysequence comprises the sequence set forth in SEQ ID NO:
 1. 7. The methodof claim 6 , wherein the regulatory sequence comprises the sequence setforth in SEQ ID NO:1 from about nucleotide −1 to about nucleotide −1148.
 8. The method of claim 6 , wherein the regulatory sequencecomprises the sequence set forth in SEQ ID NO:1 from about nucleotide −1to about nucleotide −286.
 9. The method of claim 6 , wherein theregulatory sequence comprises the sequence set forth in SEQ ID NO:1 fromabout nucleotide −1 to about nucleotide −205.
 10. The method of claim 1, wherein the contacting is by physical means.
 11. The method of claim 1, wherein the contacting is by chemical means.
 12. The method of claim 1, wherein the plant cell is selected from the group consisting ofprotoplasts, gamete producing cells, and cells which regenerate intowhole plants.
 13. The method of claim 1 , wherein said nucleic acid iscontained in a T-DNA derived vector.
 14. A plant produced by the methodof claim 1 .
 15. Plant tissue derived from a plant produced by themethod of claim 1 .
 16. A seed derived from a plant produced by themethod of claim 1 .
 17. A transformed plant cell comprising a chimericnucleic acid sequence comprising: a cycB1a;At regulatory sequenceoperably linked to a heterologous nucleic acid sequence.
 18. Thetransformed plant cell of claim 17 , wherein the regulatory sequencecomprises the sequence set forth in SEQ ID NO:1.
 19. The transformedplant cell of claim 18 , wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide −1 to aboutnucleotide −1148.
 20. The transformed plant cell of claim 18 , whereinthe regulatory sequence comprises the sequence set forth in SEQ ID NO:1from about nucleotide −1 to about nucleotide −286.
 21. The transformedplant cell of claim 18 , wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide −1 to aboutnucleotide −205.
 22. A transgenic plant comprising a heterologonsnucleic acid sequence regulated by a regulatory sequence of cycB1a;Atwherein the regulatory sequence is operably linked to the heterologousnucleic acid sequence.
 23. The transgenic plant of claim 22 , whereinthe regulatory sequence has the sequence set forth in SEQ ID NO:1. 24.The transgenic plant of claim 23 , wherein the regulatory sequencecomprises the sequence set forth in SEQ ID NO:1 from about nucleotide −1to about nucleotide −1148.
 25. The transgenic plant of claim 23 ,wherein the regulatory sequence comprises the sequence set forth in SEQID NO:1 from about nucleotide −1 to about nucleotide −286.
 26. Thetransgenic plant of claim 23 , wherein the regulatory sequence comprisesthe sequence set forth in SEQ ID NO:1 from about nucleotide −1 to aboutnucleotide −205.
 27. An isolated nucleic acid sequence comprising afunctional cycB1a;At regulatory sequence.
 28. The sequence of claim 27 ,wherein the regulatory sequence comprises the sequence set forth in SEQID NO:1.
 29. The sequence of claim 28 , wherein the regulatory sequencecomprises the sequence set forth in SEQ ID NO:1 from about nucleotide −1to about nucleotide −1148.
 30. The sequence of claim 28 , wherein theregulatory sequence comprises the sequence set forth in SEQ ID NO:1 fromabout nucleotide −1 to about nucleotide −351.
 31. The sequence of claim28 , wherein the regulatory sequence comprises the sequence set forth inSEQ ID NO:1 from about nucleotide −1 to about nucleotide −286.
 32. Thesequence of claim 28 , wherein the regulatory sequence comprises thesequence set forth in SEQ ID NO:1 from about nucleotide −1 to aboutnucleotide −205.
 33. The sequence of claim 28 , wherein the regulatorysequence comprises the sequence set forth in SEQ ID NO:1 from aboutnucleotide −1 to about nucleotide −120.
 34. The sequence of claim 28 ,wherein the regulatory sequence comprises the sequence set forth in SEQID NO:1 from about nucleotide −1 to about nucleotide −60.
 35. A nucleicacid sequence comprising a functional cycB1a;At regulatory sequence,operably linked to a nucleic acid sequence encoding a cyclin protein.36. A vector containing the nucleic acid sequence of claim 27 or 35 .37. The nucleic acid sequence of claim 35 , wherein said regulatorysequence comprises the sequence set forth in SEQ ID NO:1.
 38. Thenucleic acid sequence of claim 37 , wherein the regulatory sequencecomprises the sequence set forth in SEQ ID NO:1 from about nucleotide −1 to about nucleotide −1148.
 39. The nucleic acid sequence of claim 37 ,wherein the regulatory sequence comprises the sequence set forth in SEQID NO:1 from about nucleotide −1 to about nucleotide −351.
 40. Thenucleic acid sequence of claim 37 , wherein the regulatory sequencecomprises the sequence set forth in SEQ ID NO:1 from about nucleotide −1to about nucleotide −286.
 41. The nucleic acid sequence of claim 37 ,wherein the regulatory sequence comprises the sequence set forth in SEQID NO:1 from about nucleotide −1 to about nucleotide −205.
 42. Thenucleic acid sequence of claim 37 , wherein the regulatory sequencecomprises the sequence set forth in SEQ ID NO:1 from about nucleotide −1to about nucleotide −120.
 43. The nucleic acid sequence of claim 37 ,wherein the regulatory sequence comprises the sequence set forth in SEQID NO:1 from about nucleotide −1 to about nucleotide −60.